The present invention relates to a method and composition for treating and preventing pathogenic effects in mammals caused by intracellular calcium overload. Calcium overload occurs in the tissue and organs of mammals suffering from a disease condition associated with or resulting from insufficient tissue oxygenation. By treating the pathogenic effects of intracellular calcium overload, the present invention also effectively prevents irreversible cell damage and cell lysis in cells transiently deprived of oxygen.
Healthy cells regulate free cytosolic calcium concentrations by limiting influx of the cation across the cell""s plasma membrane, sequestering free calcium, and pumping calcium ions out of the cytosol. When a cell becomes ischemic, insufficient free energy exists to operate the ion pumps. As calcium accumulates in the cytosol, degradative enzymes become activated and begin to further affect the cell""s ability to regulate calcium. Calcium activated enzymes, e.g., phospholipases, break down the cell""s membranes, making them even xe2x80x9cleakierxe2x80x9d to calcium. Additional enzymes, e.g., proteases, also attack the molecular pumps. When oxygen is restored to the tissue, free radical oxygen species are produced that can further damage these systems.
If the combined effects of the enzymes and the free radical oxygen species becomes severe enough, the cell will not recover and maintain acceptable levels of calcium, even if it successfully re-energizes when circulation is reestablished. The cell has become irreversibly damaged and will ultimately die from an overload of calcium. This pathogenic sequence might be repeated millions of times in the first several hours following a transient interruption in blood supply to the heart or brain.
Neuron damage, following a stroke or cardiac arrest, and myocyte damage, following coronary artery occlusion, are two examples of such cell damage. When essential cellular constituents leak out of such damaged cells, the cell is referred to as lysed and, of course, is irreparable.
Various treatments have been studied for treating or preventing calcium mediated cellular damage to reduce the likelihood of cell lysis during and following a transient period of oxygen deprivation. The most common treatments involve administering chemical compounds that either limit entry of calcium ions into the cell (i.e., plasma membrane channel blockers) or antagonize the calcium activated enzymes by binding to intracellular proteins like calmodulin. These treatments, however, are not restricted to the damaged cells. They can affect the function of normal, healthy cells and cause a number of adverse side effects. More selective methods are, therefore, needed to treat or prevent calcium mediated damage in cells deprived of oxygen, while avoiding these adverse side effects.
It is an object of the present invention to treat or prevent the pathogenic effects in a mammal caused by intracellular calcium overload. It is another object to prevent irreversible cell damage or hypoxic or post-hypoxic cell lysis in a mammal that has suffered from a disease condition associated with or resulting from insufficient tissue oxygenation.
It is a more specific object of the present invention to provide methods and compositions for preventing lysis of such cells and reducing the extent of organ damage in a mammal that has suffered from anoxia, hypoxia or ischemia, such as that which occurs in cardiac arrest, pulmonary embolus, renal artery occlusion, coronary artery occlusion, occlusive stroke, hemorrhagic stroke, adult respiratory distress syndrome, neonatal respiratory distress syndrome, suffocation, or profound anemia.
Additional objects and advantages of the invention will be set forth in part in the description which follows, and in part will be apparent from the description, or may be learned by practice of the invention. The objects and advantages of the invention may be realized and obtained by means of instrumentalities and combinations particularly pointed out in the appended claims.
To achieve the objects and in accordance with the purpose of the invention, as embodied and broadly described herein, the invention comprises a method for treating or preventing pathogenic effects in a mammal caused by intracellular calcium overload comprising administering to the mammal a mixture of sodium co-transport dependent amino carboxylic acids or their physiologically acceptable salts in an amount sufficient to substantially saturate the sodium dependent amino carboxylic acid transport mechanisms of a mammalian cell""s plasma membrane. The present invention also relates to a pharmaceutical composition for treating or preventing pathogenic effects in a mammal caused by intracellular calcium overload that comprises a mixture of sodium co-transport dependent amino carboxylic acids or their physiologically acceptable salts in an amount sufficient to substantially saturate the sodium dependent transport mechanisms of a mammalian cell""s plasma membrane to treat or prevent these pathogenic effects, together with a pharmaceutically acceptable carrier.
As used in the present invention, the terms xe2x80x9csaturatexe2x80x9d and xe2x80x9csaturating concentrationsxe2x80x9d denote the accepted biochemical kinetic definition, i.e., those concentrations of substrate or ligand that maximally activate (Vmax) their respective enzyme(s), transport mechanism(s), or receptor(s).
Reference will now be made in detail to preferred embodiments of the invention.
An aspect of the present invention contemplates treating a mammal suffering with a disease condition resulting from insufficient tissue oxygenation by administrating a mixture of therapeutic amino carboxylic acid agents to alleviate the toxic effects of calcium overload on hypoxic and/or post hypoxic cells, anoxic and/or postanoxic cells, and ischemic and/or postischemic cells. The therapeutic amino carboxylic acids of the present invention can treat such cells by entering the cell""s interiors. They are referred to as sodium co-transport dependent amino carboxylic acids. To be sodium co-transport dependent means that these amino carboxylic acids are obligatorily transported into the cell in association with sodium through sodium dependent, plasma membrane transport systems.
Without being bound to any particular theory, it is believed that when the amino carboxylic acids of the present invention enter the cell with the sodium ions they cause a partial discharge of the cell membrane""s sodium electrochemical gradient. This disturbance in the gradient prevents rapid re-alkalinization of the cell, thereby inactivating pH-sensitive calcium dependent cytolytic enzymes and minimizing or eliminating the cytotoxic effects of calcium overload. All of the therapeutic amino carboxylic acid agents of the present invention are selective for identified sodium-dependent transport (xe2x80x9csymportxe2x80x9d) proteins. The sodium dependent transport systems are known to those skilled in the art to include the GLY, A, ASC, and N transporters.
In a primary embodiment of the present invention, a pharmaceutical composition is provided containing saturating concentrations of:
(a) one or more substrates for the GLY transport system;
(b) one or more substrates for the A transport system;
(c) one or more substrates for the ASC transport system;
(d) one or more substrates for the N transport system; and
(e) at least one stimulator or activator of the hepatic urea cycle.
A preferred composition of these substrates comprises a mixture of glycine, proline, histidine, serine, alanine, glutamic acid, glutamine, and arginine.
As indicated, in this embodiment, one or more administered therapeutic agents are substrates for the xe2x80x9cGLYxe2x80x9d transport system. The GLY transport system is a sodium-dependent amino carboxylic acid transport protein widely expressed in terminally differentiated mammalian cells. See H. N. Christensen, Physiological Reviews, Volume 70, p. 43-77, 1990, hereby incorporated by reference. The preferred substrate for the GLY symport is glycine.
The amino carboxylic acid substrates for the sodium dependent xe2x80x9cAxe2x80x9d transport system include metabolizable model substrates, alanine or proline, or non-metabolizable substrates, 2-aminoisobutyric acid or N-methyl-alpha-aminoisobutyric acid. The preferred substrate for the A symport is -alanine. An amino carboxylic acid substrate for the sodium dependent xe2x80x9cASCxe2x80x9d transport system is serine. Both the A and ASC transport systems are expressed in terminally differentiated mammalian cells. See, H. N. Christensen, supra.
A substrate for another sodium-dependent amino carboxylic acid transport protein, the xe2x80x9cNxe2x80x9d transport system, which expresses specific terminally differentiated mammalian cells like hepato-cytes, H. N. Christensen supra. includes amino carboxylic acids selected from histidine, asparagine, glutamic acid, or glutamine. The preferred substrate for the N symport is histidine.
Some of the amino carboxylic acids of the present invention can also stimulate the hepatic urea cycle in a mammal. This is important in situations where administration of a particular acid exceeds hyper-physiological amounts, creating potential toxicity concerns. With the hepatic urea cycle stimulating amino carboxylic acids of the invention, however, one can advantageously increase dosage amounts of the other amino carboxylic acids, in some cases above the hyper-physiological amounts. The preferred hepatic urea cycle stimulating amino carboxylic acid is arginine.
Additional therapeutically active agents can be co-administered with these sodium co-transport dependent amino carboxylic acids of the present invention. Examples of classes of agents that may complement the action of the therapeutic amino carboxylic acids of the present invention include:
(1) a plasma volume expander, such as dextran;
(2) a water soluble magnesium salt, such as magnesium chloride;
(3) a thrombolytic enzyme, such as streptokinase;
(4) scavengers of toxic oxygen metabolites, such as superoxide dismutase and/or catalase;
(5) a xanthine oxidase inhibitor, such as allopurinol; and
(6) a substance which binds free iron, such as desferrioxamine.
The amino carboxylic acids contemplated for use in the present invention can be obtained in various ways, including hydrolysis of a protein or fermentation of glucose, for example. Amino acids have also been created in the laboratory by passing an electric discharge through a mixture of ammonia, methane, and water vapor. Hawley, xe2x80x9cThe Condensed Chemical Dictionary,xe2x80x9d 10th Ed., 1981, pp. 48-49.
The amino carboxylic acids of the present invention can be administered in intravenous infusions after a hypoxic, anoxic, or ischemic condition occurs to treat or prevent irreversible cell injury and cell lysis caused by intracellular calcium overload. For example, the amino carboxylic acids of the present invention can be administered by intravenous infusion immediately after a cerebral infarction, a myocardial infarction, asphyxia, or cardiopulmonary arrest. Alternatively, the amino carboxylic acids can be administered by intravenous or intra-arterial infusion concurrently and in association with thrombolytic therapy.
The therapeutic agents of the present invention can be used prophylactically in surgical settings where circulation to an organ or organ system is deliberately interrupted, e.g. coronary artery bypass surgery, tissue grafting, endarterectomy, angioplasty, etc. The present invention also contemplates adding the amino carboxylic acids to a cardioplegia solution for organ perfusion, and to perfusion and preservative solutions for organ transplantation.
The amino carboxylic acids of the present invention can be administered in mixtures with one another, or together with a physiologically suitable carrier or vehicle. If appropriate, the aramino carboxylic acids may be administered in the form of a physiologically acceptable salt, for example, an acid addition salt. A preferred carrier is 5% dextrose in water or half-strength normal saline, buffered to pH 7.4 with a physiologically acceptable buffer substance.
In administering the amino carboxylic acids of the present invention, a loading dose is given at the start of treatment. Thereafter, a maintenance dose is to be administered either continuously or intermittently in order to maintain optimal levels of the amino carboxylic acids in the blood. The timing and amount of the maintenance dose can be determined by intermittently monitoring the levels of the amino carboxylic acids in the blood.
The amount of amino carboxylic acids administered as a loading or maintenance dose will depend upon the particular acids employed, the number of acids administered, and the method of application. Due to the potentially toxic effects of hyper-physiological concentrations of amino carboxylic acids in the blood, they are typically administered so as to attain a blood plasma concentration of no greater than 200 to 300 milligrams total free amino carboxylic acid per deciliter (100 ml) of blood. As mentioned above, however, if an hepatic cycle stimulating amino carboxylic acid of the present invention is co-administered, it is possible to increase the dosage amount.
In accordance with a preferred embodiment of the present invention, there is provided a mixture of glycine, proline, histidine, serine, alanine, glutamic acid, glutamine, and arginine administered by intravenous infusion into a 70 kg mammal. In this embodiment, the acids can be dissolved in one liter of a suitable water based carrier to a concentration of between 0.5-0.7% glycine (0.6% being preferred), between 0.6-0.9% proline (0.8% being preferred), between 0.8-1.2% histidine (1.0% being preferred), between 0.5-0.8% serine (0.7% being preferred), between 0.5-0.7% alanine (0.6% being preferred), between 0.3-0.5% glutamic acid (0.4% being preferred), between 0.3-0.5% glutamine (0.4% being preferred), and between 0.4-0.6% arginine (0.5% being preferred). A large volume loading dose of from about 2 to 3 ml per kg of body weight is first administered over a period of 30 minutes. Thereafter, a maintenance dose of about 1 to 2 ml per kg of body weight per hour is administered for 4 to 6 hours. Further administration of the therapeutic solution is determined by monitoring the blood amino carboxylic acid levels in order to maintain a total blood concentration of total amino carboxylic acids of between 200 to 300 mg per deciliter (100 ml) of blood. Treatment can continue for at least 12 hours after tissue oxygenation is normalized.
The above embodiment (corrected for the effect of dilution in total body water) has been demonstrated to achieve near or substantial saturation of the sodium-dependent amino carboxylic acid transporters of the hepatocyte plasma membrane. Analogous concentrations of these eight constituent amino carboxylic acids were employed in the xe2x80x9cMixturexe2x80x9d described in Examples 2-7 below.
A particularly preferred embodiment, described in Example 8, comprises a mixture of 8 amino carboxylic acids formulated as a dry powder that is to be reconstituted as a buffered intravenous solution with 5% dextrose (or half strength normal saline). The amino acids comprising this mixture are those whose entry into mammalian cells is mediated by one or more of the sodium-dependent plasma membrane transport systems: alanine, proline, histidine, serine, glycine, glutamine, glutamic acid, and arginine. In this embodiment, the following amino carboxylic acids can be dissolved by gram amounts in one liter of a suitable water-based carrier: between 7 g to 34 g alanine (between 20 g to 30 g being preferred, and 30 g particularly preferred); between 9 g to 42 g proline (between 28 g to 40 g being preferred, and 35 g particularly preferred); between 8 g to 28 g histidine (between 10 g to 24 g being preferred, and 15 g particularly preferred); between 8 g to 34 g serine (between 14 g to 30 g being preferred and 20 g particularly preferred); between 7 g to 38 g glycine (between 15 g to 35 g being preferred, and 32 g particularly preferred); between 5 g to 34 g glutamine (between 14 g to 30 g being preferred, and 21 g particularly preferred); between 1 g to 9 g glutamic acid (between 2 g to 6 g being preferred, and 4 g particularly preferred); and between 6 g to 58 g arginine (between 30 g to 52 g being preferred, and 43 g particular preferred).
The relatively high concentrations of the eight amino carboxylic acids of the particularly preferred embodiment (described above) are designed to fully saturate the sodium-dependent transport mechanisms not only of hepatocytes, but also of essentially all other mammalian cells considered to be susceptible to calcium overload. An initial dosing of the particular preferred embodiment is about 1.0 gm/Kg of body weight given over 60 minutes, and the maintenance dose is about 0.2 gm/Kg/hour for a period of 6 to 12 hours or longer as clinical implications warrant.
The suitability of the therapeutic acids of the present invention for treating or preventing irreversible cell damage and cell lysis caused by intracellular calcium overload can be predicted from the following examples.